Abstract: Maize flour is limited in its food industry applications owing to the imbalance in its nutritional composition and low bioavailability of some of its critical nutrients. To improve the nutritional quality of maize flour, its fermentation with Bacillus subtilis was optimized in this study using orthogonal tests, and the fermentation effects were analyzed using starch and protein as nutritional indicators. As a result, when using the yield and soluble solids and amino acid nitrogen contents as indicators, the optimal fermentation conditions were as follows: material-to-liquid ratio of 1:1, inoculum volume of 19%, ammonium sulfate addition of 2%, and fermentation for 60 h at 37 ℃. Under these conditions, the amylose, amylopectin, and starch levels in the maize flour decreased by 4.12%, 3.93%, and 3.99%, respectively, whereas the amounts of reducing sugars, soluble proteins, and crude proteins increased by 23.62%, 16.76%, and 3.63%, respectively. Compared with the corresponding values in the control, the estimated glycemic index decreased to 55.67 and the amount of slowly digestible starch increased by 76.00%. In conclusion, fermentation with Bacillus subtilis may decrease the digestibility of maize flour starch, which has promising benefits for maintaining blood glucose levels. Moreover, the increase in soluble protein content and decrease in amylopectin amount after fermentation with Bacillus subtilis indicate that this treatment improves the nutritional quality of maize flour.